An experiment was conducted to validate and optimize the procedure of spot urine sampling with urinary creatinine as a marker to estimate urine outputs of dairy cows. Twelve lactating cows were used in a randomized complete block design. Cows were grouped and randomly assigned to 2 experimental diets: a corn silage-based diet and an alfalfa silage-based diet with supplemental potassium. The experiment lasted for 21 d and total collection (TC) of urine was conducted for the last 3 d. Twelve spot samples of urine from individual cows were collected over a 3-d period during TC to represent every 2-h sampling in a 24-h cycle. Creatinine excretion rate (mg/kg of body weight per d) was variable among cows from 16.7 to 34.5 with an average of 27.3. Creatinine concentrations of spot samples within cow were averaged to simulate urine samples obtained from various spot sampling frequencies (equally spaced 12, 6, 4, and 2 time points starting at feeding: 12TP, 6TP, 4TP, and 2TP, respectively). Large diurnal variation of urinary creatinine concentration was observed within cow. Creatinine concentration was greater (75 vs. 65 mg/dL) for 12TP compared with TC, resulting in underestimating (29.8 vs. 32.6 kg/d) urine outputs. When compared among 12TP, 6TP, 4TP, and 2TP, creatinine concentrations were different and urine outputs tended to be different for 2TP compared with 12TP, 6TP, and 4TP. In addition, despite underestimation of urine output, a regression analysis indicated strong linear relationships between 12TP, 6TP, or 4TP and TC, suggesting that this technique can successfully identify the differences in urine outputs altered by dietary treatments. However, 4TP failed to detect statistical differences in urine outputs between a corn silage-based diet and the alfalfa silage-based diet with supplemental potassium, indicating that a spot urine sampling frequency of at least 6 was required to identify dietary effects on urine outputs. According to the pattern of diurnal changes in urinary creatinine concentration, a spot sample at about 10 h after feeding may have potential to obtain a urine sample that is more representative (i.e., creatinine concentration) of TC urine compared with urine from multiple sampling frequencies. Overall, urinary creatinine as a marker with spot sampling of urine underestimated urine output. However, 12TP and 6TP were successful in identifying changes in urine outputs by dietary treatments.

Indirect methods of spot sampling with intrinsic markers to estimate fecal output and nutrient digestibility often have been used in dairy nutrition research as alternatives to total collection of feces (TC) because of labor and expense. However, fecal output and nutrient digestibility estimated from the indirect method must be accurate regardless of altering dietary conditions. This experiment was designed to validate the accuracy of using indigestible neutral detergent fiber (iNDF) or acid-insoluble ash (AIA) as intrinsic markers to estimate fecal outputs and nutrient digestibility compared with TC and to determine the optimal number of spot sampling events to accurately determine fecal output and then nutrient excretion. The experiment used 12 multiparous lactating Holstein cows in a randomized complete block design. Cows were blocked by days in milk and milk yield and randomly assigned to 1 of 2 diets: a diet containing about 49% corn silage on a dry matter basis and a diet containing about 48% alfalfa silage with high by-product (soyhulls) and supplemental K. During the final 3 d of 21-d periods, TC was performed, and 12 spot samples were collected for the same 3 d to represent every 2 h in a 24-h cycle. Fecal outputs and nutrient digestibility of dry matter, organic matter, or nitrogen estimated with iNDF or AIA as an intrinsic marker were compared with TC. Overall, fecal outputs and digestibility estimated with iNDF were similar to that estimated with TC, whereas AIA overestimated fecal output by 44 to 61% and underestimated nutrient digestibilities by 16 to 32%. However, potential differences in statistical inference of dietary effects between iNDF and TC were found. Data from individual spot samples were aggregated to represent spot sampling frequencies of 12 (SP12), 6 (SP6), 4 (SP4), or 2 (SP2) evenly spaced events starting at feeding time. Compared with TC, SP12 produced similar fecal content of iNDF, organic matter, and nitrogen, but fecal AIA content was greater. Furthermore, compared with SP12, SP6 produced similar fecal content of all nutrients, whereas marker and nutrient concentrations in SP4 and SP2 were different. In this experiment, iNDF was a better fecal marker than AIA, and a spot sampling frequency of at least 6 events was necessary. However, interpretation of dietary effects could be confounded when iNDF was used to estimate fecal outputs.

Three experiments assessed branched-chain volatile fatty acid (BCVFA) stimulation of neutral detergent fiber (NDF) disappearance after 24 h of incubation in batch cultures derived from ruminal fluid inocula that were enriched with particulate-phase bacteria. In experiment 1, a control was compared with 3 treatments with isomolar doses of all 3 BCVFA (plus valerate), all 3 branched-chain AA (BCAA), or half of each BCVFA and BCAA mix with either alfalfa or grass hays (50%) and ground corn grain (50%). A portion of the BCAA and BCVFA doses were enriched with 13C, and valerate (also enriched with 13C) was added with BCVFA. Although BCAA yielded a similar production of BCVFA compared with dosing BCVFA, equimolar substitution of BCVFA for BCAA decreased the percentage of N in bacterial pellets when alfalfa hay was fed but increased N when grass hay was fed. Substituting BCVFA for BCAA increased total fatty acid (FA) concentration with alfalfa hay. Dosing of BCAA or BCVFA did not affect total branched-chain FA, iso-FA, or anteiso-FA percentages in bacterial total FA, whereas numerous individual FA isomers and their 13C enrichments were affected by these treatments. Increasing recovery of the 13C dose from respective labeled BCVFA primers indicated facilitated BCVFA uptake and incorporation into FA compared with BCAA, whereas increased recovery of 13C from labeled BCAA in the bacteria pellet but not in the FA fraction suggested direct assimilation into bacterial protein. The BCVFA and valerate were dosed in varying combinations that either summed to 4 mM (experiment 2) or had only 1 mM no matter what combination (experiment 3). In general, grass hay was more responsive to stimulation in NDF digestibility by BCVFA than was alfalfa hay, which was attributed to the higher degradable protein in the latter. The net production of the BCVFA (after subtracting dose) was affected by source and combination of BCVFA. Isovalerate dosing tended to increase its own net production; in contrast, isobutyrate seemed to be used more when it was added alone, but 2-methylbutyrate seemed to be preferred over isobutyrate when 2-methylbutyrate was added. Results supported potential interactions, including potential feedback in production from feed BCAA or increased concentration-dependent competition for dosed BCVFA into cellular products. Under our conditions, the BCVFA appear to be more readily available than BCAA, probably because of regulated BCAA transport and metabolism. Valerate consistently provided no benefit. Using nonparametric ranking, all 3 BCVFA or either isovalerate or isobutyrate (both yielding iso-FA) should be combined with 2-methylbutyrate (yielding anteiso-FA) as a potential opportunity to improve NDF digestibility when rumen-degraded BCAA are limited in diets to decrease environmental impact from N in waste.

An experiment was conducted to examine effects of prepartum, postpartum, or continuous prepartum and postpartum supply of rumen-protected lysine (RPLys) and rumen-protected methionine (RPMet) on performance and blood metabolites of transition cows. The experiment consisted of a prepartum (3 wk), postpartum (3 wk), and carryover (10 wk) period. Eighty-eight prepartum cows (36 primiparous and 52 multiparous cows) were blocked by parity and expected calving date and assigned to 1 of 4 treatments arranged factorially. Treatments were a prepartum diet (12% crude protein on a dry matter basis) without (Pre-) or with supplemental RPLys (10 g of digestible Lys/cow per day) and RPMet (4 g of digestible Met/cow per day; Pre+) followed by postpartum diets (16% crude protein on a dry matter basis) without (Post-) or with supplemental RPLys (26 g of digestible Lys/cow per day) and RPMet (11 g of digestible Met/cow per day; Post+). Prepartum, only 2 treatments were applied, but postpartum cows received treatments of Pre-Post-, Pre-Post+, Pre+Post-, or Pre+Post+. During the prepartum period, treatment did not affect dry matter intake and body weight. During the postpartum period, milk protein content was greater (3.23 vs. 3.11%) for Post+ compared with Post- independent of prepartum treatment. However, dry matter intake, body weight, milk yield, and yields of milk components were not affected by Post+ versus Post-. No effects of prepartum treatment or interactions between pre- and postpartum treatments were observed on postpartum performance of cows. No effects of pre- and postpartum supplementation of RPLys and RPMet on performance during the carryover period were found except prepartum supplementation of RPLys and RPMet decreased somatic cell count (4.60 vs. 4.83; log10 transformed) compared with Pre- in the postpartum period and this effect continued during the carryover period [i.e., 4.42 and 4.55 (log10 transformed) for Pre+ and Pre-, respectively]. Prepartum supplementation of RPLys and RPMet increased or tended to increase plasma concentration of Lys, Met, and branched-chain AA compared with Pre- in prepartum cows. Cows on Post+ tended to have greater plasma Lys concentration compared with Post-, but plasma Met concentration was not affected. Health events of postpartum cows were not affected by treatments. In conclusion, we did not observe positive effects of supplementing with RPLys and RPMet on performance of prepartum and postpartum cows. However, prepartum supply of RPLys and RPMet may have potential to improve udder health and immune status of fresh cows.

To support improving genetic potential for increased milk production, intake of digestible carbohydrate must also increase to provide digestible energy and microbial protein synthesis. We hypothesized that the provision of exogenous branched-chain volatile fatty acids (BCVFA) would improve both neutral detergent fiber (NDF) degradability and efficiency of microbial protein synthesis. However, BCVFA should be more beneficial with increasing efficiency of bacterial protein synthesis associated with increasing passage rate (k_p). We also hypothesized that decreasing pH would increase the need for isobutyrate over 2-methylbutyrate. To study these effects independent from other sources of variation in vivo, we evaluated continuous cultures without (control) versus with BCVFA (0 vs. 2 mmol/d each of isobutyrate, isovalerate, and 2-methylbutyrate), low versus high k_p of the particulate phase (2.5 vs. 5.0%/h), and high versus low pH (ranging from 6.3 to 6.8 diurnally vs. 5.7 to 6.2) in a 2 × 2 × 2 factorial arrangement of treatments. Diets were 50% forage pellets and 50% grain pellets administered twice daily. Without an interaction, NDF degradability tended to increase from 29.7 to 35.0% for main effects of control compared with BCVFA treatments. Provision of BCVFA increased methanogenesis, presumably resulting from improved NDF degradability. Decreasing pH decreased methane production. Total volatile fatty acid (VFA) and acetate production were decreased with increasing k_p, even though true organic matter degradability and bacterial nitrogen flow were not affected by treatments. Decreasing pH decreased acetate but increased propionate and valerate production, probably resulting from a shift in bacterial taxa and associated VFA stoichiometry. Decreasing pH decreased isobutyrate and isovalerate production while increasing 2-methylbutyrate production on a net basis (subtracting doses). Supplementing BCVFA improved NDF degradability in continuous cultures administered moderate (15.4%) crude protein diets (excluding urea in buffer) without major interactions with culture pH and k_p.

An experiment was conducted to examine effects of supplemental lysophospholipids (LPL) in dairy cows. Eight ruminally cannulated lactating Holstein cows were used in a replicated 4 × 4 Latin square design. Dietary treatments were (1) a dairy ration [CON; 55% forage and 45% concentrate on a dry matter (DM) basis], (2) a positive control diet supplemented with monensin (MON; 16 mg/kg in dietary DM; Elanco Animal Health, Greenfield, IN], (3) a control diet supplemented with low LPL (0.05% of dietary DM; Lipidol Ultra, Easy Bio Inc., Seoul, South Korea), and (4) a control diet supplemented with high LPL (0.075% of dietary DM). Experimental periods were 21 d with 14-d diet adaptation and 7-d sample collection. Daily intake and milk yield were measured and rumen contents were collected for fermentation characteristics and bacterial population. Spot urine and fecal samples (8 samples/cow per period) were collected to determine nutrient digestibility and dietary N utilization. All data were analyzed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC; group and cow within group were random effects and treatments, time, and their interaction were fixed effects). Preplanned contrasts were made to determine effect of MON versus CON, effect of LPL versus MON, and linear effect of increasing LPL. In the current study, responses to MON generally agreed with effects of monensin observed in the literature (increased milk yield and feed efficiency but decreased milk fat content). Supplementation of LPL to the diet did not alter DM intake but linearly increased milk yield, resulting in increases in feed efficiency (milk yield/DM intake) and milk protein and fat yields. However, total-tract digestibility of DM and organic matter tended to be lower (60.9 vs. 62.2% and 61.8 vs. 63.1%, respectively) for LPL compared with CON. Linear increases in milk N secretion and decreases in urinary N excretion were observed with increasing LPL in the diet. A slight decrease in acetate proportion in the rumen for LPL was found. Relative to MON, very few bacteria in the rumen were affected with increasing LPL. In conclusion, LPL is a potential feed additive that can increase milk yield and components and dietary N utilization. However, more studies with large numbers of animals are needed to confirm the effect of LPL on production. Similar positive effects on production were observed between LPL and MON, but individual mechanisms were likely different according to ruminal fermentation characteristics. Further studies are needed to explore the mode of action of LPL in dairy cows.